An immunosuppressive or immune excluded tumor microenvironment (TME) plays a key role in limiting the response of many tumor types to immunotherapy. One attractive strategy to accomplish increased lymphocyte infiltration in tumors is the use of cytokines, which can directly impact multiple immune pathways and reprogram the TME to enable a robust immune response against cancer cells. Unfortunately, despite this obvious potential, many cytokines have been limited clinically due to toxicity concerns. Rational drug delivery strategies that can rescue the therapeutic potential of cytokines could act as an important step in our ability to carefully manipulate the anti-tumor immune response in the TME and open the door for more effective immunotherapies. Nanoparticles (NPs) are a promising vehicle for the rescue of toxic cytokines. While many studies have used NPs to improve efficacy and toxicity, there remains a substantial knowledge gap surrounding the role of NP biophysical properties on enhanced delivery. There is much that is not yet understood about how nanoparticles traffic and how these differences can affect therapeutic outcomes. We are uniquely positioned to investigate the role of NP biophysical properties on cytokine delivery given our extensive experience in both NP design for targeted tumor cell delivery and in polyelectrolyte layer-by-layer (LbL) assembly. LbL-NP systems can be designed to modulate the release of multiple drugs from the core and from surrounding layers, often with time dependent staged release; whereas, manipulating the outer layer to possess certain surface chemistries and targeting moieties can significantly impact trafficking of particles on both the anatomical and cellular level. Using this system will allow for a systematic investigation of the role of these unique NP properties on effective cytokine delivery. The goal of this work is to understand and control the delivery of cytokines against solid tumors using LbL-NPs as a tool, with a focus on the impact of trafficking, localization and release kinetics of the particle and payload. Our work will focus on interleukin-12 (IL-12), one of the most potent and toxic proinflammatory cytokines for which we have recently demonstrated improved efficacy and lowered systemic toxicity by using LbL-NPs that bind to the surface membrane of ovarian cancer cells. Our studies will take place within the context of advanced serous ovarian cancer (OC), which has shown limited response to existing immunotherapies, and non-small cell lung cancers (NCSLC), which is highly responsive, but only for a defined subset of patients. IL-12 loaded NPs with external layers possessing a range of surface chemistries and targeting moieties will be examined for cellular and subcellular uptake and immune cell stimulation. Cytokine release kinetics will be examined and optimized, and nanoparticle systems will be examined in vivo for delivery of cytokines alone and in combination with anti- PD1 treatments in orthotopic syngeneic animal models. 1